Use of a bacillus thuringiensis microbe for controlling lesser mealworm, alphitobius diaperinus

ABSTRACT

The subject invention concerns the use of a novel and useful bioinsecticide against the lesser mealworm (Alphitobius diaperinus). The lesser mealworm is a devastating pest in the poultry industry. The bioinsecticide of the subject invention is a novel B. thuringiensis microbe referred to as B.t. PS40D1, or mutants thereof. The spores or toxin crystals of this microbe are useful to control the lesser mealworm in various environments.

CROSS-REFERENCE TO A RELATED APPLICATION

This is a continuation-in-part of co-pending application Ser. No.07/155,189, filed Feb. 12, 1988, now U.S. Pat. No. 4,999,192.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (B.t.) produces an insect toxin designated asδ-endotoxin. It is synthesized by the B.t. sporulating cell. The toxin,upon being ingested in its crystalline form by susceptible insectlarvae, is transformed into biologically active moieties by the insectgut juice proteases. The primary target is insect cells of the gutepithelium, which are rapidly destroyed.

The reported activity spectrum of B.t. covers insect species within theorder Lepidoptera, many of which are major pests in agriculture andforesty. The activity spectrum also includes the insect order Diptera,which includes mosquitos and black flies. See Couch, T. L. (1980)"Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,"Developments in Industrial Microbiology 22:61-76; Beegle, C. C., (1978)"Use of Entomogenous Bacteria in Agroecosystems," Developments inIndustrial Microbiology 20:97-104. U.S. Pat. No. 4,910,016 discloses aB.t. strain active against coleopteran and lepidopteran pests. U.S. Pat.No. 4,849,217 discloses a B.t. strain which is active against theEgyptian alfalfa weevil.

The lesser mealworm (Alphitobius diaperinus) is a cosmopolitan pest ofhouseholds, stored grain facilities, and poultry houses. It usuallyfeeds on damp and moldy grain, milled products, and spoiled foods. Dueto significant growth in the poultry industry in recent years, thisinsect has become one of the major pest species present in the litterand structural components of poultry houses. The larvae of A. diaperinusingest chicken feed and other organic matter (including dead or moribundchicks), are reservoirs for a multitude of pathogens which are threatsto poultry production, and cause extensive structural damage to poultryhouses by tunneling into the insulation and soft wood of the facility.The loss of insulation due to the holes and tunnels bored by A.diaperinus larvae results in greater heating costs as well as poorerfeed conversion efficiency by poultry due to the lack of adequatetemperature control in the poultry houses. Additionally, since poultrywill feed on these insects as an alternative food source, they mayexperience lesser weight gains than if fed their normal nutrientbalanced diet. The larvae of A. diaperinus have also been implicated incausing lesions on poultry and may increase their susceptibility todisease due to dust stirred up by the birds while they are scratchingfor insects. A. diaperinus may also cause allergic reactions in humans.

Current control techniques are inadequate for controlling this pest andwill not eradicate the pest problem. They include the following: 1)thoroughly cleaning the poultry house and having it remain empty for aprolonged period of time; 2) applying insecticides to the structure andfloors of the house after it is cleaned; 3) using insect growthregulators; and 4) using steinernematid and heterorhabditid nematodes asbeetle entomopathogens. These methods are ineffective and expensive.Thus, there is an urgent need for the effective control of the lessermealworm.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns the use of a novel Bacillus thuringiensis(B.t.) isolate. This novel B.t. isolate, known herein as Bacillusthuringiensis PS40D1 (B.t. PS40D1), has been shown, unexpectedly andadvantageously, to be active against the pest known as the lessermealworm (Alphitobius diaperinus).

The subject invention also includes the use of mutants of B.t. PS40D1which have substantially the same pesticidal properties as B.t. PS40D1.Procedures for making mutants are well known in the microbiological art.Ultraviolet light and nitrosoguanidine are used extensively toward thisend.

Further, the invention also includes the use of treated cells ofsubstantially intact B.t. PS40D1 cells, or mutants thereof, to prolongthe pesticidal activity when the substantially intact cells are appliedto the environment of the lesser mealworm. Such treatment can be bychemical or physical means, or a combination of chemical or physicalmeans, so long as the technique does not deleteriously affect theproperties of the pesticide, nor diminish the cellular capability inprotecting the pesticide. The treated B.t. PS40D1 cell acts as aprotective coating for the pesticidal toxin. The toxin becomes availableto act as such upon ingestion by the lesser mealworm.

DETAILED DISCLOSURE OF THE INVENTION

The novel Bacillus thuringiensis isolate of the subject invention ischaracterized in U.S. application Ser. No. 07/155,189, filed on Feb. 12,1988, now U.S. Pat. No. 4,999,192. That characterization is incorporatedherein by reference to said application.

The culture disclosed in this application has been deposited in theAgricultural Research Service Patent Culture Collection (NRRL), NorthernRegional Research Center, 1815 North University Street, Peoria, Ill.61604, U.S.A.

    ______________________________________                                        Culture          Repository No.                                                                            Deposit date                                     ______________________________________                                        Bacillus thuringiensis PS40D1                                                                  NRRL B-18300                                                                              Feb. 3, 1988                                     ______________________________________                                    

The subject culture has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.The deposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least thirty (30)years after the data of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

B.t. PS40D1, NRRL B-18300, can be cultured using standard art media andfermentation techniques. Upon completion of the fermentation cycle, thebacteria can be harvested by first separating the B.t. spores and toxincrystals from the fermentation broth by means well known in the art. Therecovered B.t. spores and/or toxin crystals can be formulated into awettable powder, spray, liquid concentrate, granules, or otherformulations by the addition of surfactants, dispersants, inert carriersand other components to facilitate handling and application forparticular target pests. These formulation and application proceduresare all well known in the art. Since the B.t. spores and/or toxincrystals of the invention are non-toxic to humans or animals, the use ofexcess amounts of such is not detrimental. It is well within the skillof those in this art to use levels of the B.t. spores and/or toxincrystals which provide optimum pesticidal results with minimum costs.

Formulated products can be sprayed or applied onto areas inhabited bythe lesser mealworm.

Another approach that can be taken is to incorporated the spores andtoxin crystals of B.t. PS40D1 into bait granules containing anattractant and applying these granules to the soil for control of thelesser mealworm.

The B.t. PS40D1 cells can be treated prior to formulation to prolong thepesticidal activity when the cells are applied to the environment of thelesser mealworm. Such treatment can be by chemical or physical means, orby a combination of chemical and/or physical means, so long as thetechnique does not deleteriously affect the properties of the pesticide,nor diminish the cellular capability in protecting the pesticide.Examples of chemical reagents are halogenating agents, particularlyhalogens of atomic no. 17-80. More particularly, iodine can be usedunder mild conditions and for sufficient time to achieve the desiredresults. Other suitable techniques include treatment with aldehydes,such as formaldehyde and glutaraldehyde; anti-infectives, such aszephiran chloride; alcohols, such as isopropyl and ethanol; varioushistologic fixatives, such as Bouin's fixative and Helly's fixative(See: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman andCompany, 1967); or a combination of physical (heat) and chemical agentsthat prolong the activity of the toxin produced in the cell when thecell is applied to the environment of the target pest(s). Examples ofphysical means are short wavelength radiation such as gamma-radiationand X-radiation, freezing, UV irradiation, lyophilization, and the like.

The gene(s) from the novel B.t. isolate of the subject invention can beintroduced into other microbes. The toxin(s) expressed by the gene(s)can be used to control the lesser mealworm. Such toxins can beformulated by standard techniques into suitable sprays and, also,incorporated into poultry feed.

A wide variety of ways are known and available for introducing the B.t.gene(s) expressing the toxin into the microorganism host underconditions which allow for stable maintenance and expression of thegene. The transformants can be isolated in accordance with conventionalways, usually employing a selection technique, which allows forselection of the desired organism as against unmodified organism ortransferring organisms, when present. The transformants then can betested for lesser mealworm activity.

Suitable host cells, where the cells will be treated to prolong theactivity of the toxin in the cell when the then treated cell is appliedto the environment of the lesser mealworm, may include eitherprokaryotes or eukaryotes, normally being limited to those cells whichdo not produce substances toxic to higher organisms, such as mammals.However, organisms which produce substances toxic to higher organismscould be used, where the toxin is unstable or the level of applicationsufficiently low as to avoid any possibility of toxicity to a mammalianhost. As hosts, of particular interest will be the prokaryotes and thelower eukaryotes, such as fungi. Illustrative prokaryotes, bothGram-negative and -positive, include Enterobacteriaceae, such asEscherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae;Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium,Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter;Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, suchas Phycomycetes and Ascomycetes, which includes yeast, such asSaccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such asRhodotorula, Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t. gene intothe host, availability of expression systems, efficiency of expression,stability of the toxin in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as amicrocapsule include protective qualities for the toxin, such as thickcell walls, pigmentation, and intracellular packaging or formation ofinclusion bodies; leaf affinity; lack of mammalian toxicity;attractiveness to pests for ingestion; ease of killing and fixingwithout damage to the toxin; and the like. Other considerations includeease of formulation and handling, economics, storage stability, and thelike.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., and the like. Specific organismsinclude Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomycescerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis,and the like.

The transformed host microbe containing the B.t. gene(s) of the subjectinvention can be used, if desired, without treatment, as describedabove, to control the lesser mealworm.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Culturing B.t. PS40D1, NRRL B-18300

A subculture of B.t. PS40D1, NRRL B-18300 can be used to inoculate thefollowing medium, a peptone, glucose, salts medium.

    ______________________________________                                        Bacto Peptone          7.5    g/l                                             Glucose                1.0    g/l                                             KH.sub.2 PO.sub.4      3.4    g/l                                             K.sub.2 HPO.sub.4      4.35   g/l                                             Salt Solution          5.0    ml/l                                            CaCl.sub.2 Solution    5.0    ml/l                                            Salts Solution (100 ml)                                                       MgSO.sub.4.7H.sub.2 O  2.46   g                                               MnSO.sub.4.H.sub.2 O   0.04   g                                               ZnSO.sub.4.7H.sub.2 O  0.28   g                                               FeSO.sub.4.7H.sub.2 O  0.40   g                                               CaCl.sub.2 Solution (100 ml)                                                  CaCl.sub.2.2H.sub.2 O  3.66   g                                               pH 7.2                                                                        ______________________________________                                    

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and/or toxin crystals, obtained in the abovefermentation, can be isolated by procedures well known in the art. Afrequently-used procedure is to subject the harvested fermentation brothto separation techniques, e.g., centrifugation.

EXAMPLE 2 Testing of B.t. PS40D1, NRRL B-18300 Spores and Toxin CrystalsAgainst the Lesser Mealworm

B.t. PS40D1, NRRL B-18300 spores and toxin crystals were tested againstthe lesser mealworm. The assay was conducted as follows:

Lesser mealworm first or second instar larvae were exposed to apreparation of B.t. PS40D1 on a feeding substrate consisting of apoultry feed slurry. The assay was incubated for 6 days and themortality assessed by noting dead/alive larvae. Each test was replicatedseveral times.

B.t. PS40D1 was found to be active in the above test. The percentmortality was 60.

In view of the above test result, B.t. PS40D1 (isolate and/or toxin) canbe used to control the lesser mealworm populations through severalmechanisms: 1) as a feed through in poultry where the residual in thepoultry feces would be toxic to the mealworm (the material could beplaced in the chicke feed, water, mineral/vitamin supplement, on incombination with other therapeutic agents); 2) as a surface spray whichcould be used to treat structural components, insulation, litter,floors, cages, or other areas where beetles reside and feed; and 3) as abait spray or formulation where the beetles would be attracted to thefood substrate or attractant, eat the B.t. PS40D1 treated bait and dieor become moribund or less fit, thus reducing the populations of thebeetles.

The use of this biocontrol agent is advantageous because it isenvironmentally benign, and B.t. microbes, as a class, are well knowneffective biological control agents for other pest species. They providelong term population control of insects without consideration for toxicor residual toxicity effects attributed to the current syntheticchemicals which are utilized.

Since there is no known way to predict which insects the subject B.t.isolate may be active against, it is necessary to conduct tests againsteach insect species in order to determine whether there is activityagainst such insect. For example, tests are presently being conductedagainst dermestid beetles, for example, Dermestes maculatus andDermestes lardarius, which are also found in poultry houses. Thus, workis continuing on the subject B.t. isolate to determine whether it or itstoxins can be used to control other insects.

We claim:
 1. A process for controlling the lesser mealworm whichcomprises contacting said pest, or the environment of said pest, with aneffective amount of Bacillus thuringiensis PS40D1 spores or toxincrystals, having the identifying characteristics of NRRL B-18300, ormutants thereof.
 2. A process, according to claim 1, wherein said insectpest is contacted with an insect-controlling sufficient amount ofBacillus thuringiensis PS40D1 spores or toxin crystals, or mutantsthereof, by incorporating said Bacillus thuringiensis PS40D1 into apoultry feed.
 3. A process, according to claim 1, wherein said insectpest is contacted with an insect-controlling sufficient amount ofBacillus thuringiensis PS40D1 spores or toxin crystals, or mutantsthereof, by incorporating said Bacillus thuringiensis PS40D1 into asurface spray.
 4. A process, according to claim 1, wherein said insectpest is contacted with an insect-controlling sufficient amount ofBacillus thuringiensis PS40D1 spores or toxin crystals, or mutantsthereof, by incorporating said Bacillus thuringiensis PS40D1 into a baitspray or formulation.
 5. A process, according to claim 1, whereinsubstantially intact Bacillus thuringiensis PS40D1 cells, or mutantsthereof, are treated to prolong the pesticidal activity when thesubstantially intact cells are applied to the environment of the lessermealworm.
 6. A process for controlling the lesser mealworm whichcomprises contacting said pest, or the environment of said pest, with aneffective amount of a toxin(s) obtained from Bacillus thuringiensisPS40D1, having the identifying characteristics of NRRL B-18300, ormutants thereof.